1. Field of the Invention
The present invention relates to a method of preparing an integrated spot microarray by forming a pattern on a formed spot using a photocatalyst, a microarray and a microarray substrate manufactured according to the method.
2. Description of the Related Art
A DNA microarray (also called a DNA chip) can be used to detect a biomolecule (DNA, RNA, or the like) existing in a sample from a living body. Using a DNA microarray, several hundreds through tens of thousands of biomolecules can be detected and nucleotide sequences thereof can be determined in parallel. On a DNA microarray, a high density of DNA detection points (spots )are regularly arranged on a glass substrate or a silicon substrate of a determined size, usually from several square centimeters to several tens of square centimeters. Immobilized to each spot is a single-stranded nucleic acid of known sequence (e.g., a gene fragment) that can function as a probe (detector). Generally, microarrays with high spot density are preferred since they provide the capability of detecting more biomolecules in parallel in one reaction.
Detection of a biomolecule (e.g. DNA) using a microarray is achieved when a biomolecule complementary to an immobilized probe polynucleotide hybridizes with the probe and is thus retained on the microarray surface. For example, an aqueous solution containing fluorescent-labeled nucleic acids can be applied to the surface of a microarray to let the nucleic acids hybridize to complementary probes on the microarray surface. The solution is then removed and only nucleic acids hybridized to the probes on the microarray surface are retained after the procedure. By detecting fluorescence of the fluorescent material labeling the nucleic acids hybridized to probes on the DNA microarray, it can be determined whether any nucleic acids complementary to the probes were present in the sample nucleic acids.
DNA microarrays can be classified into two kinds based on the method of manufacturing the microarray: a photolithography type and a spotting type.
The photolithography type is prepared by synthesizing oligonucleotides on a substrate using a photolithography method used in a manufacturing process for a semiconductor integrated circuit. The resulting DNA microarray can have a high density of DNA detection points. Illustrative methods for manufacturing such polynucleotide microarrays are disclosed in U.S. Pat. Nos. 5,744,305 and 5,445,934.
In contrast, the spotting type is prepared by spotting droplets of a solution containing a prepared probe oligonucleotide on the surface of a substrate (e.g. an aminosilane-coated substrate). The oligonucleotides can covalently attach to the substrate when dried. Illustrative methods for manufacturing such polynucleotide microarrays are disclosed in U.S. Pat. No. 5,87,522.
These two kinds of DNA microarrays have different properties. DNA microarrays made by the photolithography method ensure a high measuring sensitivity and reproducibility, and can be used in analyzing a single nucleotide polymorphism (SNP). However, the method and resultant microarrays are expensive since a mask for each base needs to be made prior to the synthesis and each mask is expensive to make. For example, 80 masks are required when a probe having 20 bases is synthesized. Thus, only a few research institutions can afford to use DNA microarrays made using the photolithography method.
Microarrays manufactured by the spotting method are inexpensive since the method only involves oligonucleotide solutions and a substrate. However, the resulting DNA microarrays have lower density and uniformity of DNA immobilized on the substrate compared to those made by the photolithography method. For example, recently marketed microarrays manufactured by the photolithography method have a spot size of about 5 μm; while those manufactured by the spotting method have a minimum spot size of about 60 μm (SMP2 pin). As a result, microarrays manufactured by the spotting method have a lower spot density than those manufactured by the photolithography method.
Accordingly, there is a need to reduce spot size to make microarrays with high spot density. The present invention provides methods to address the need.